The Candida antarctica lipase B (CALB) is able to catalyze synthesis reactions, with esterification reactions catalyzed by CALB being particularly well-studied. While CALB is able to react with a wide variety of alcohol substrates to form esters, it is more limited in the type of acid substrate it recognizes, with preference for straight-chain fatty acids.
Eukaryotic hosts like fungi and the yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Pichia pastoris have been engineered to produce a secreted form of CALB. CALB is also supplied commercially, expressed and secreted in a recombinant fungal or yeast host (Novozymes and cLecta product data sheets). However, low transformation efficiency and long growth periods make these eukaryotic systems difficult to use for high-throughput screening of large numbers of enzyme variants. Typically, DNA constructs must pass or “shuttle” through an E. coli or other bacterial host prior to introduction into the eukaryotic host, and the cells must be grown for several days, often in the presence of an inducer to stimulate expression. In addition, a generally useful secretion system for extracellular lipase expression in yeast is lacking, and efficient recovery of the recombinant lipase requires the lipase to be active outside the host cell in a cell-free system. The expression of the eukaryotic CALB enzyme has been accomplished in the common bacterial (prokaryotic) host E. coli, but not secretion, and so a subsequent cell-lysis step is required to liberate the lipase for characterization. Frequently only the hydrolytic activity of these recombinant lipases was confirmed, but not synthesis activity. A recent publication summarizes the difficulty of expressing CALB in a heterologous host, especially a bacterial host (Larsen et al., 2008). The authors hypothesize that incorrect protein folding in E. coli is a limitation in expression of CALB in this bacterial host.
A CALB variant with improved activity for synthesis reactions would improve the efficiency of esterification, amidation and transesterification reactions and permit the economic manufacture of compounds using an enzyme catalyst. A CALB variant with improved activity for synthesis reactions would also permit the use of an enzyme catalyst to synthesize derivatives of hindered substrates. While some CALB variants having improved hydrolytic activity have been prepared, these variants are irrelevant to improving synthetic activity, which occurs in the absence of water. In order to identify amino acid changes in the native CALB sequence to target for change and to measure and understand the impact of amino acid changes on structure and function of the enzyme, it is also necessary to devise methods for predicting the protein structure in synthesis conditions, that is in the absence of water, and also to devise a method for expressing the enzyme variants and isolating them in a form suitable for synthesis reactions.